Aminopropanol derivatives

ABSTRACT

Compounds of formula I: 
                         
wherein R 1 , R 2 , n and m are as defined in the specification, processes for their production, their uses and pharmaceutical compositions containing them.

This is a divisional of application Ser. No. 10/569,696 filed on Feb.27, 2006, which is National Stage of International Application No.PCT/EP04/09589 filed on Aug. 27, 2004, which claims benefit of GreatBritain Application No. 0320196.9 filed on Aug. 28, 2003, and GreatBritain Application No. 0324206.2 filed on Oct. 15, 2003, the entiredisclosures of which are hereby incorporated by reference.

The present invention relates to organic compounds, a process for theirproduction and pharmaceutical compositions containing them.

More particularly the present invention provides a compound of formulaI:

whereineach of m and n, independently, is 1, 2 or 3;X is O or a direct bond;R₁ is

-   -   a phenylalkyl wherein alkyl is a straight- or branched        (C₆₋₂₀)carbon chain; or    -   a phenylalkyl wherein alkyl is a straight- or branched        (C₁₋₃₀)carbon chain wherein said phenylalkyl is substituted at        the phenyl residue by    -   a straight- or branched (C₆₋₂₀)carbon chain optionally        substituted by halogen,    -   a straight- or branched (C₆₋₂₀)alkoxy chain optionally        substituted by halogen,    -   a straight- or branched (C₆₋₂₀)alkenyloxy,    -   phenylalkoxy, halophenylalkoxy, phenylalkoxyalkyl, phenoxyalkoxy        or phenoxyalkyl,    -   cycloalkylalkyl substituted by C₆₋₂₀alkyl,    -   heteroarylalkyl substituted by C₆₋₂₀alkyl,    -   heterocyclic C₆₋₂₀alkyl or    -   heterocyclic alkyl substituted by C₂₋₂₀alkyl,        and wherein        the alkyl moiety may have    -   in the carbon chain, a bond or a heteroatom selected from a        double bond, a triple bond, O, S, sulfinyl, sulfonyl, or NR₅,        wherein R₅ is H, alkyl, aralkyl, acyl or alkoxycarbonyl, and    -   as a substituent alkoxy, alkenyloxy, alkynyloxy, aralkyloxy,        acyl, alkylamino, alkylthio, acylamino, alkoxycarbonyl,        alkoxycarbonylamino, acyloxy, alkylcarbamoyl, nitro, halogen,        amino, hydroxy or carboxy, and        R₂ is

-   -   wherein each of R₃ and R₄, independently, is H or C₁₋₄alkyl,        wherein alkyl is optionally substituted by 1, 2 or 3 halogen        atoms;

In free form or in salt form.

Halogen is F, Cl, Br or I. Alkyl or alkoxy may be straight or branchedchain.

Cycloalkyl is preferably C₃₋₁₀cycloalkyl, more preferably C₃₋₆cycloalkyland includes, for example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl or cycloheptyl.

Acyl may be a residue R_(y)—CO— wherein R_(y) is C₁₋₆alkyl,C₃₋₆cycloalkyl, phenyl or phenyl-C₁₋₄alkyl.

When in the compounds of formula I the carbon chain as R₁ issubstituted, it is preferably substituted by halogen, nitro, amino,hydroxy or carboxy. When the carbon chain is interrupted by anoptionally substituted phenylene, the carbon chain is preferablyunsubstituted. When the phenylene moiety is substituted, it ispreferably substituted by halogen, nitro, amino, methoxy, hydroxy orcarboxy.

In the compounds of the invention, the following significances arepreferred individually or in any sub-combination:

-   1. m and n are each 1 or 2, preferably 1.-   2. X is O.-   3. R₁ is C₁₃₋₂₀alkyl, optionally substituted by nitro, halogen,    amino, hydroxy or carboxy, and, more preferably those wherein R₁ is    phenylalkyl substituted by C₆₋₁₄-alkyl chain optionally substituted    by halogen and the alkyl moiety is a C₁₋₆alkyl optionally    substituted by hydroxy. More preferably, R₁ is phenyl-C₁₋₆alkyl,    e.g. phenyl-C₁₋₆alkyl, e.g. phenyl-C₂alkyl, substituted on the    phenyl by a straight or branched, preferably straight, C₆₋₁₄alkyl    chain. The C₆₋₁₄alkyl chain may be in ortho, meta or para,    preferably in para.-   4. each of R₃ and R₄ is H.

A particularly preferred compound is phosphoric acidmono-[2-hydroxymethyl-4-(4-octyl-phenyl)-2-(1oxo-1,3-dihydro-isoindol-2-yl)-butyl]ester.

Compounds of formula I may exist in free form or in salt form, e.g.addition salts with e.g. inorganic acids, such as hydrochloride,hydrobromide or sulfate, salts with organic acids, such as acetate,fumarate, maleate, benzoate, citrate, malate, methanesulfonate orbenzenesulfonate salts; when R₃ or R₄ is H, R₂ may also be present insalt form, e.g. an ammonium salt or salts with metals such as sodium,potassium, calcium, zinc or magnesium, or a mixture thereof. Compoundsof formula I and their salts in hydrate or solvate forms are also partof the invention.

The compounds of formula I have one or more asymmetric centers in themolecule, and thus various optical isomers may be obtained. The presentinvention also encompasses enantiomers, racemates, diastereoisomers andmixtures thereof. The central asymmetric carbon atom may have the R or Sconfiguration. Moreover, when the compounds of formula I includegeometric isomers, the present invention embraces cis-compounds,trans-compounds and mixtures thereof. Similar considerations apply inrelation to starting materials exhibiting asymmetric carbon atoms orunsaturated bonds as mentioned above.

A compound of formula I may be prepared by reacting a compound offormula II:

wherein m, n, X, R₁ and R₂ are as defined in formula 1;with an aromatic 1,2-dicarbaldehyde, e.g. benzene-1,2-dicarbaldehyde,and recovering the resulting compound of formula I in free or salt form.

The process may be performed according to methods known in the art, e.g.as described in the examples.

A compound of formula II (e.g. a racemic mixture thereof) may beobtained as described in WO 02/18395 or WO 02/076995.

The present invention also provides a compound of formula I or formulaII, wherein greater than 70% by weight of the compound is in the form ofthe S enantiomer, or greater than 70% by weight of the compound is inthe form of the R enantiomer, e.g. greater than 90% is in the form ofthe R or S enantiomer. More preferably greater than 95% by weight, e.g.greater than 99% by weight of the compound is in the form of the R or Senantiomer. Thus the invention may relate to the substantially pure R orS enantiomer (e.g. the S enantiomer substantially free of the Renantiomer or vice versa), preferably the S enantiomer, of a compound offormula 1 or formula II. Particularly preferred are the substantiallypure (e.g. greater than 99% by weight) R or S enantiomers, especiallythe S enantiomers, of phosphoric acidmono-[2-amino-2-hydroxymethyl-4-(4-octyl-phenyl)-butyl]ester(FTY720-phosphate) and phosphoric acidmono-[2-hydroxymethyl-4-(4-octyl-phenyl)-2-(1-oxo-1,3-dihydro-isoindol-2-yl-butyl]ester.

Compounds having the following 3-dimensional configuration are generallypreferred:

Enantiomers of the compounds of formula I and II cannot besatisfactorily separated by standard methods. According to the presentinvention, separation of the enantiomers is achieved by the use of novelseparation techniques and synthesis strategies.

A compound of formula I, wherein greater than 70% by weight of thecompound is in the form of the R or S enantiomer, e.g. the substantiallypure R or S enantiomer, may be obtained by:

a) separation of the S enantiomer from the R enantiomer in a racemicmixture of a compound of formula I, using chromatography on a chiralstationary phase; or

b) reacting a compound of formula II, wherein greater than 70% by weightof the compound is in the form of the R or S enantiomer, e.g. thesubstantially pure R or S enantiomer of a compound of formula II, withan aromatic 1,2-dicarbaldehyde e.g. benzene-1,2-dicarbaldehyde.

According to method a), the chromatographic separation is preferablycarried out using a chiral ion-exchange phase based on quinine carbamateor quinidine carbamate as chiral selector, e.g. a quinine carbamatephase (8S,9R) available commercially under the tradename ProntoSILChiral AX QN-1:

A compound of formula II, wherein greater than 70% by weight of thecompound is in the form of the R or S enantiomer, may be obtained bydeprotecting a compound of formula III, wherein greater than 70% byweight of the compound of formula III is in the form of the R or Senantiomer:

wherein m, n, X, R₁ and R₂ are as defined above and R₆ is an aminoprotecting group, and, where required, converting the compounds offormula I obtained in free form into the desired salt form, or viceversa.

Examples of suitable amino protecting groups as R₆ are e.g. as disclosedin “Protective Groups in Organic Synthesis” T. W. Greene, J. Wiley &Sons NY, 2^(nd) ed., chapter 7, 1991, and references therein, e.g. acyl,e.g. tert.-butoxy-carbonyl, benzyloxycarbonyl, 9-fluorenyl methoxycarbonyl, trifluoroacetyl, trimethylsilyethanesulfonyl and the like.

Alternatively and more preferably a compound of formula II, whereingreater than 70% by weight of the compound is in the form of the R or Senantiomer, may be obtained by deprotecting a compound of formula IIIaor IIIb, wherein greater than 70% by weight of the compound of formulaIIIa or IIIb is in the form of the R or S enantiomer:

wherein n, m, X, R₁ and R₂ are as defined above and R₆′ is asimultaneous OH and amino protecting group, e.g. such that R₆′ togetherwith the O and N atoms to which it is attached, the asymmetric carbonatom and 1 to 3 further carbon atoms forms a cyclic residue, e.g. a 5 to7-membered heterocyclic ring, e.g oxazolidin-2-one (in IIIa R₆′ is—C(O)—) or 2-methyl-4,5-dihydro-oxazole (in IIIb R₆′ is —C(CH₃)—).

The removal of the R₆ or R₆′ protecting group in a compound of formulaIII, IIIa or IIIb may conveniently be performed according to methodsknown in the art, e.g. by hydrolysis, e.g. in a basic medium, forexample using a hydroxide such as barium hydroxide. It may also beperformed by hydrogenolysis, e.g. in the presence of Pearlman'scatalyst, e.g. as disclosed in J. Org. Chem., 1998, 63, 23752377.

Thus in a further alternative aspect the present invention provides acompound of formula III, IIIa of IIIb as defined above, in free or saltform. The compounds of formula III, IIIa or IIIb have one or moreasymmetric centers in the molecule, and thus various optical isomers maybe obtained. The present invention also encompasses enantiomers,racemates, diastereoisomers and mixtures thereof.

The removal of the R₆ or R₆′ protecting group in a compound of formulaIII, IIIa or IIIb may conveniently be performed according to methodsknown in the art, e.g. by hydrolysis, e.g. in a basic medium, forexample using a hydroxide such as barium hydroxide. It may also beperformed by hydrogenolysis, e.g. in the presence of Pearlman'scatalyst, e.g. as disclosed in J. Org. Chem., 1998, 63, 2375-2377.

A compound of formula III, IIIa or IIIb comprising greater than 70% byweight of the R or S enantiomer may be obtained by separating the Senantiomer from the R enantiomer in a racemic mixture of a compound offormula III, IIIa or IIIb, using chromatography (MPLC. HPLC, SFC) orsimulated moving bed (multi-column) chromatography with apolysaccharide-based chiral stationary phase, preferably an amylose-typephase, e.g. amylose tris[(S)-α-methylbenzyl carbamate coated on silicagel substrate, as available under the tradename CHIRALPAK AS and shownbelow, or in an immobilized form as prepared according to the processesdescribed in WO 97/04011 and WO 97/49733:

A compound of formula III, IIIa or IIIb comprising greater than 70% byweight of the R or S enantiomer may alternatively be obtained byremoving the hydrolysable groups present in R₂′ in a compound of formulaIV, IVa or IVb comprising greater than 70% by weight of the R or Senantiomer:

wherein m, n, X, R₁, R₆ and R₆′ are as defined above and R₂′ is

wherein each of R₃′ and R₄′ is a hydrolysable group.

Preferably R₃′ and R₄′ are identical and have the significance of e.g.phenyl or benzyl or form together a cyclic system such as in1,5-dihydro-2,4,3-benzodioxaphosphepin.

A compound of formula IV, IVa or IVb comprising greater than 70% byweight of the R or S enantiomer may be obtained by separating the Senantiomer from the R enantiomer in a racemic mixture of a compound offormula IV, IVa or IVb, e.g. as described above for the separation ofenantiomers of compounds of formula III, IIIa or IIIb.

A compound of formula IV, IVa or IVb, e.g. a racemic mixture thereof,wherein X is O may be obtained by reacting a compound of formula V, Vaor Vb:

wherein m, n, R₁, R₆ and R₆′ are as defined above,with a phosphorylating agent, e.g. a phosphorochloridate, e.g.diphenylchlorophosphate or dibenzylchlorophosphate, cyanoethylphosphate,a phosphoramidate such as N-phenyl phosphoramidate,3-(diethylamino)-1,5-dihydro-2,4,3-benzodioxaphosphepin and the like.The reaction may be carried out according to methods known in the art,e.g. as disclosed in J. Org. Chem. supra. In the compounds of formulaIIIa the amino group is preferably in protected form, as R′₄ when R₄ isother than acyl.

A compound of formula IV, IVa or IVb, e.g. a racemic mixture thereof,wherein X is a direct bond may be obtained by reacting a compound offormula V′, Va′ or Vb′:

wherein m, n, R₁, R₆ and R₆′ are as defined above, andY is a leaving group, e.g. Br,with a phosphorylating agent, e.g. diethyl phosphite under reducingconditions, e.g. in the presence of NaH. The reaction may be performedin accordance with methods known in the art.

Alternatively the chiral separation may be performed at an earlier stagein the process. Thus a compound of formula IV, IVa or IVb comprisinggreater than 70% by weight of the R or S enantiomer may be obtained byreacting a compound of formula V, Va, Vb, V′, Va′ or Vb′ comprisinggreater than 70% by weight of the R or S enantiomer with aphosphorylating agent. A compound of formula V, Va, Vb, V′, Va′ or Vb′comprising greater than 70% by weight of the R or S enantiomer may beobtained by separating the S enantiomer from the R enantiomer in aracemic mixture of a compound of formula V, Va, Vb, V′, Va′ or Vb′, e.g.using HPLC or simulated moving bed (multi-column) chromatography with apolysaccharide based chiral stationary phase as described above forseparation of the enantiomers of a compound of formula IV, IVa or IVb.

A compound of formula V or V′ may be prepared by reacting a compound offormula VI:

wherein m, n and R₁ are as defined above and R₉ is OH or a leavinggroup, e.g. Br,with an amino protecting group donor compound. A compound of formula Va,Va′, Vb or Vb′ may be prepared by reacting a compound of formula VI withan OH and amino protecting compound, e.g the OH and amino protection maybe performed simultaneously by reacting the free aminoalcohol oraminodiol of formula VI in order to obtain a cyclic residue, e.g. a 5 to7-membered heterocyclic ring, e.g oxazolidin-2-one or2-methyl-4,5-dihydro-oxazole, e.g. by reaction with Cbo-Cl,Boc-anhydride, triethylortho acetate and acetonitrile, or phosgene underbasic conditions.

At any stage in the process, R₁ in any of the formulae above mayoptionally be converted to an alternative R₁ group using known methods.For example, a compound of formula Vb wherein R₁ is(4-benzyloxy-phenyl)-ethyl may be converted to an alternative compoundof formula Vb wherein R₁ is [4-(6-fluoro-hexyloxy)-phenyl]-ethyl by (a)removal of a benzyl group from R₁ by hydrogenation to leave a(4-hydroxy-phenyl)-ethyl residue, followed by (b) reaction with1-bromo-6-fluorohexane. The alternative compound of formula Vb may thenundergo chiral separation or be converted to a compound of formula IVbas described above.

The compounds of formulae III, IIIa, IIIb, IV, IVa and IVb comprisinggreater than 70% by weight of the R or S enantiomer used as startingmaterials, and salts thereof are also novel and form part of the presentinvention.

In a further alternative aspect, a compound of formula II whereingreater than 70% by weight of the compound is in the form of the R or Senantiomer, may be obtained by deprotecting and hydrolyzing a compoundof formula VII, wherein greater than 70% by weight of the compound is inthe form of the R or S enantiomer

wherein m, n, X and R₁ are as defined above, and R₂″ is

wherein each of R₃′ and R₄′ is a hydrolysable group, e.g. tert-butyl,and R₇ is an amino protecting group, e.g. benzyloxycarbonyl.

A compound of formula VII, wherein greater than 70% by weight of thecompound is in the form of the R or S enantiomer, may be obtained byreacting a compound of formula VIII, wherein greater than 70% by weightof the compound is in the form of the R or S enantiomer,

with a phosphorylating agent, e.g. as described above.

Insofar as the production of the starting materials is not particularlydescribed, the compounds are known or may be prepared analogously tomethods known in the art or as disclosed in the Examples hereinafter.

The following Examples are illustrative of the invention.

-   RT=room temperature-   CBO=benzyloxycarbonyl-   FTY720=2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol-   EDTA=ethylenediaminetetraacetic acid-   OPA=ortho-phthalaldehyde (benzene-1,2-dicarbaldehyde)

EXAMPLE 1 Phosphoric Acidmono-[(R/S)-2-hydroxymethyl-4-(4-octyl-phenyl)-2-(1-oxo-1,3-dihydro-isoindol-2-yl)-butyl]ester

a) (R/S)-4-Hydroxymethyl-4-[2-(4-octyl-phenyl)-ethyl]-oxazolidin-2-one

Benzyl chloroformate (0.45 ml; 3.2 mmol) is added to a suspension ofFTY720.HCl (1.03 g, 3 mmol) in 2N NaOH (20 ml). The reaction is kept atRT over night and in order to complete the reaction further benzylchloroformate (0.9 ml; 6.4 mmol) is added. After 2 days at RT thereaction is acidified with 1N HCl, extracted with methylenechloride andpurified on a silica gel column using methylenechloride/methanol/aceticacid_(50%) (9/1/0.125) as mobile phase.

[M+H]⁺: 334 (ESI-MS)

b)(R/S)-4-[2-(4-Octyl-phenyl)-ethyl]-4-(3-oxo-1,5-dihydro-3lambda*5*-benzo[e][1,3,2]dioxaphosphepin-3-yloxymethyl)-oxazolidin-2-one

To a solution of the endproduct of a) (2.4 g; 7.2 mmol) inmethylenechloride/THF 1/1 (100 ml) at 0° C. is added tetrazole(recrystallized; 2.52 g; 36 mmol) and3-(diethylamino)-1,5-dihydro-2,4,3-benzodioxaphosphepintriphenyl-phosphite(5.17 g; 21.6 mmol). After 18 hours at RT, H₂O₂ (8.2 ml [30% in water];72 mmol) is added (cooling) to the solution and kept at RT foradditional 90 minutes. After quenching with saturated Na₂S₂O₃ solution(100 ml) the reaction is extracted with ethylacetate (three times). Theorganic layer is dried over Na₂SO₄ and the compound is purified onsilica gel using cyclohexane/ethylacetate 1/1 as mobile phase.

c) Phosphoric Acidmono-{(R/S)-4-[2-(4-octyl-phenyl)-ethyl]-2-oxo-oxazolidin-4-ylmethyl}ester

The endproduct of step b) (1.03 g; 2 mmol) is hydrogenated at normalpressure (Pd/C_(10%); 50 mg) over a period of 90 minutes. Afterfiltration the reaction is concentrated and used in step d) withoutfurther purification.

d) Phosphoric Acidmono-[(R/S)-2-amino-2-hydroxymethyl-4-(4-octyl-phenyl)-butyl]ester((R/S-FTY720-phosphate)

To a solution of the endproduct of step c) in ethanol (20 ml) LiOH (20ml; 10% solution in water) is added. After 24 hours at reflux thereaction is neutralized with HCl (1N in water) and concentrated. Theresidue is treated with glacial acetic acid (5 ml) and precipitation ofthe endproduct occurs after addition water (50 ml). After filtering,washing (water) and drying pure endproduct is obtained without anyfurther purification.

e) Phosphoric Acidmono-[(R/S)-2-hydroxymethyl-4-(4-octyl-phenyl)-2-(1-oxo-1,3-dihydro-isoindol-2-yl)-butyl]ester(OPA-Derivatization)

The endproduct of step d) ((R/S)-FTY720-phosphate) (50 mg; 0.125 mmol)is suspended in a solution of EDTA (0.5 ml; 10 mM in water) and aqueousboric acid (0.5 ml; 3% in water; adjusted to pH 10.5 with aqueousKOH_(10%)). After addition of OPA (33 mg, 0.25 mmol), dissolved inethanol (0.5 ml), the reaction is kept at RT for 1 hour (ultrasound).After that the pH is adjusted to 3.5 (aqueous HCl; 1N) and extractedwith ethylacetate (three times). The organic layer is dried over Na₂SO₄and the compound is purified on silica gel usingmethylenechloride/methanol (95/5→0/100) as mobile phase.

[M−H]⁻: 502.5 (ESI-MS)

EXAMPLE 2 Phosphoric Acidmono-[(R)-2-hydroxymethyl-4-(4-octyl-phenyl)-2-(1-oxo-1,3-dihydro-isoindol-2-yl)-butyl]ester

Example 2 is obtained after separation of the final product of step b)by HPLC on CHIRALPAK AS column at a preparative scale (ethanol/n-hexane40/60 as mobile phase), or by simulated moving bed chromatography onHPLC columns packed with immobilized amylose tris[(S)-α-methylbenzylcarbamate coated on silica gel (n-hexane/ethanol/chloroform 60/20/20 asthe mobile phase; feed concentration, 1%) and applying steps c), d) ande) as described for example 1.

EXAMPLE 3 Phosphoric Acidmono-[(S)-2-hydroxymethyl-4-(4-octyl-phenyl)-2-(1-oxo-1,3-dihydro-isoindol-2-yl)-butyl]ester

Example 3 is obtained after separation of the final product of step b)by HPLC on CHIRALPAK AS column at a preparative scale (ethanol/n-hexane40/60 as mobile phase), or by simulated moving bed chromatography onHPLC columns packed with immobilized amylose tris[(S)-α-methylbenzylcarbamate coated on silica gel (n-hexane/ethanol/chloroform 60/20/20 asthe mobile phase; feed concentration, 1%) and applying steps c), d) ande) as described for example 1.

EXAMPLE 4((R/S)-4-{2-[4-(6-Fluoro-hexyloxy)-phenyl]-ethyl}-2-methyl-4,5-dihydro-oxazol-4-yl)-methanol

a) To a solution of 4-(2-hydroxy-ethyl)-phenol (50 g, 0.36 mol) inethanol (400 ml) is added potassium carbonate (75 g, 0.54 mol, 1.5 eq)and benzyl bromide (47.2 ml, 0.39 mol, 1.1 eq), the reaction mixture isstirred at RT overnight. The reaction mixture is then filtered offthrough celite and concentrated under vacuum.2-(4-Benzyloxy-phenyl)-ethanol is isolated after crystallization withdiethyl ether.

b) To a solution of 2-(4-benzyloxy-phenyl-ethanol (78.72 g, 0.34 mol) inmethylene chloride (400 ml) is added triethylamine (67.3 ml, 0.44 mol,1.4 eq), then at 0° C. is added mesylchloride (34.8 ml, 0.44 mol, 1.3eq). The reaction mixture is stirred at 0° C. for 30 minutes and allowedto rise to RT. The reaction mixture is extracted with methylene chloride(2×300 ml), the combined organic layers are then washed with brine(2×300 ml) and concentrated under vacuum.

c) To the crude product in solution in ethyl acetate (600 ml) is addedsodium iodide (67.2 g, 0.44 mol, 1.3 eq) and the reaction mixture isstirred under reflux for 6 hours. After filtration, the organic layer iswashed with brine (3×400 ml), dried with Na₂SO₄, filtered andconcentrated under vacuum. 1-Benzyloxy-4-(2-iodo-ethyl)-benzene isisolated after crystallization with diethyl ether.

d) To a solution of acetamidomalonate (59.4 g, 0.27 mol, 2 eq) in drydimethylformamide (400 ml) is added at 0° C. under inert atmospheresodium hydride (60% in oil) (9.94 g, 0.49 mol, 1.8 eq), the reactionmixture is stirred for 3 hours at 0° C.1-Benzyloxy-4-(2-iodo-ethyl)-benzene (46.8 g, 0.13 mol, 1 eq) insolution in dry dimethylformamide (250 ml) is then slowly added at 0° C.and the reaction mixture is stirred at RT overnight. The reactionmixture is quenched with few drops of methanol and concentrated almostto dryness under vacuum, then extracted with ethyl acetate and washedsubsequently with 1N HCl (2×500 ml), saturated solution of NaHCO₃ (2×500ml) and brine (2×500 ml), dried with Na₂SO₄, filtered and concentratedunder vacuum. 2-Acetylamino-2-[2-(4-benzyloxy-phenyl)-ethyl]-malonicacid diethyl ester is isolated after multiple crystallization usingdiethyl ether.

e) To a solution of2-acetylamino-2-[2-(4-benzyloxy-phenyl)-ethyl]-malonic acid diethylester (44.1 g, 0.1 mol) in ethanol water (2/1) (285 ml/285 ml) is addedCaCl₂ (28.5 g, 0.26 mol, 2.5 eq) and NaBH₄ by portion (19.4 g, 0.52 mol,5.0 eq), the reaction mixture is stirred overnight at RT. At 0° C. thereaction mixture is carefully quenched with drop wise methanol (10 ml)and concentrated to almost dryness under vacuum. The crude mixture isextracted with ethyl acetate (4×500 ml) and washed subsequently with 1NHCl (2×300 ml), saturated solution of NaHCO₃ (2×300 ml) and brine (2×300ml). The combined organic layers are then dried with Na₂SO₄, filteredand concentrated under vacuum.N-[3-(4-Benzyloxy-phenyl)-1,1-bis-hydroxymethyl-propyl]-acetamide iscarried on without further purification.

f) To a solution of crudeN-[3-(4-benzyloxy-phenyl)-1,1-bis-hydroxymethyl-propyl]-acetamide in amixture of tetrahydrofuran, methanol, water (1/2/2) (450 ml/1900 ml 1900ml) is added at RT lithium hydroxide (32.7 g, 1.36 mol, 8.0 eq). Thereaction mixture is stirred at 65° C. for 5 hours, then extracted withethyl acetate (500 ml) and washed with brine (2×300 ml), the combinedorganic layers are then dried with Na₂SO₄, filtered and concentratedunder vacuum. 2-Amino-2-[2-(4-benzyloxy-phenyl)-ethyl]-propane-1,3-diolis isolated after crystallization using ethyl acetate.

g) To a solution of2-amino-2-[2-(4-benzyloxy-phenyl)-ethyl]-propane-1,3-diol (31.1 g, 0.10mol) in acetonitrile (2.38 l) is added triethylortho acetate (17.1 ml,0.12 mol, 1.2 eq) and acetic acid (5.48 ml, 0.11 mol, 1.1 eq), thereaction mixture is then stirred at 80° C. for 5 hours. The reactionmixture is then concentrated under vacuum,{4-[2-(4-benzyloxy-phenyl)-ethyl]-2-methyl-4,5-dihydro-oxazol-4-yl}-methanolis isolated after crystallization with ethyl acetate.

h) To a solution of{4-[2-(4-benzyloxy-phenyl)-ethyl]-2-methyl-4,5-dihydro-oxazol-4-yl}-methanol(26.1 g, 0.08 mol) in methanol (800 ml) is added palladium on charcoal(2.6 g, 10% wt), and the reaction mixture is stirred under hydrogenatmosphere at RT for 5 hours. The reaction mixture is then filteredthrough celite and concentrated under vacuum.(R/S)-4-[2-(4-Hydroxymethyl-2-methyl-4,5-dihydro-oxazol-4-yl)-ethyl]-phenolis isolated in quantitative yield after crystallization with ethylacetate and hexanes.

i) To a solution of(R/S)-4-[2-(4-hydroxymethyl-2-methyl-4,5-dihydro-oxazol-4-yl)-ethyl]-phenol(500 mg, 2.12 mmol) in dry DMF (8 ml) is added under inert atmosphereCs₂CO₃ (901 mg, 2.76 mmol, 1.3 eq.) and 1-bromo-6-fluorohexane (464.1mg, 2.55 mmol, 1.2 eq.). The reaction mixture is stirred under inertatmosphere at 85° C. overnight. A saturated solution of NaHCO₃ (20 ml)and ethyl acetate (40 ml) are then added. The organic layer is separatedand the aqueous phase is extracted with ethyl acetate (3×40 ml). Thecombined organic extracts are washed with brine and 1M HCl, dried overMgSO₄, and evaporated to dryness. Purification by flash chromatography(cy Hexane/ethyl acetate (9/1) to (1/1) and (0/1)) affords(R/S-(4-{2-[4-(6-fluoro-hexyloxy)-phenyl]-ethyl}-2-methyl-4,5-dihydro-oxazol-4-yl)-methanolas colorless oil.

Separation of enantiomers of((R/S)-4-{2-[4-(6-Fluoro-hexyloxy)-phenyl]-ethyl}-2-methyl-4,5-dihydro-oxazol-4-yl)-methanolis performed by HPLC on CHIRALPAK AD column at a preparative scale(n-hexane/2-propanol 96/6 as the mobile phase).

EXAMPLE 5 Phosphoric Acidmono-{(S)-2-amino-4-[4-(6-fluoro-hexyloxy)-phenyl]-2-hydroxymethyl-butyl}ester

a) To a solution of chiral((S)-4-{2-[4-(6-fluoro-hexyloxy)-phenyl]-ethyl}-2-methyl-4,5-dihydro-oxazol-4-yl)-methanol(300 mg, 0.80 mmol) and tetrazole (337.4 mg, 4.82 mmol, 6 eq.,recrystallized from toluene) in dry THF (6 ml) at −25° C. is added3-diethylamino-1,5-dihydro-benzo[e][1,3,2]dioxaphosphepine (433.5 μL,1.56 mmol, 1.95 eq.). The reaction mixture is stirred under argon at−25° C. for 3 h, then allowed to come back to RT. Then, H₂O₂ (30%, 75μL, 4.0 mmol, 5 eq.) is injected at 0° C. with vigorous stirring. Thereaction mixture is stirred for further 30 min, followed by addition ofsaturated sodium thiosulfate solution (1 ml). The organic layer isseparated and the aqueous phase is extracted with ether (3×20 ml). Thecombined organic extracts are washed with brine, dried over MgSO₄, andevaporated to dryness. Purification by flash chromatography (ethylacetate) affords phosphoric acid di-tert-butyl ester(S)-4-{2-[4-(6-fluoro-hexyloxy)-phenyl]-ethyl}-2-methyl-4,5-dihydro-oxazol-4-ylmethylester as colorless oil.

b) To a solution of phosphoric acid di-tertbutyl ester(S)-4-{2-[4-(6-fluoro-hexyloxy)-phenyl]-ethyl}-2-methyl-4,5-dihydro-oxazol-4-ylmethylester (33 mg, 0.050 mmol) in ethanol (2 ml) is added conc. HCl (2 ml).The reaction mixture is stirred at 85° C. for 2 hours, then concentratedto dryness. The residue is re-dissolved in ethyl acetate andprecipitated with hexanes. The solid is filtered off, washed with dryether and dried under vacuum to afford phosphoric acidmono-{(S)-2-amino-4-[4-(6-fluoro-hexyloxy)-phenyl]-2-hydroxymethyl-butyl}esteras a colorless powder.

EXAMPLE 6 Phosphoric Acidmono-{(R)-2-amino-4-[4-(6-fluoro-hexyloxy)-phenyl]-2-hydroxymethyl-butyl}ester

The synthesis is performed applying the chemistry described for example5.

EXAMPLE 7 Phosphoric Acidmono-[(S)-4-[4-(6-fluoro-hexyloxy)-phenyl]-2-hydroxymethyl-2-(1-oxo-1,3-dihydro-isoindol-2-yl)-butyl]ester

OPA-derivatization is performed according to the procedure given inexample 1, step e.

EXAMPLE 8 Phosphoric Acidmono-[(R)-4-[4-(6-fluoro-hexyloxy)-phenyl]-2-hydroxymethyl-2-(1-oxo-1,3-dihydro-isoindol-2-yl)-butyl]ester

OPA-derivatization is performed according to the procedure given inexample 1, step e.

EXAMPLE 9 Phosphoric Acidmono-((R)-2-amino-4-{4-[2-(3-fluoro-phenoxy)-ethoxy]-phenyl}-2-hydroxymethyl-butyl)ester

To a solution of 4-(2-hydroxy-ethyl)-phenol (50 g, 0.36 mol) in ethanol(400 ml) is added potassium carbonate (75 g, 0.54 mol, 1.5 eq) andbenzyl bromide (47.2 ml, 0.39 mol, 1.1 eq), the reaction mixture isstirred at room temperature overnight. The reaction mixture is thenfiltered off trough celite and concentrated under vacuum.2-(4-Benzyloxy-phenyl)-ethanol is isolated after crystallization withdiethyl ether (82.6 g, 95%).

To a solution of 2-(4-benzyloxy-phenyl)-ethanol (78.72 g, 0.34 mol) inmethylene chloride (400 ml) is added triethylamine (67.3 ml, 0.44 mol,1.4 eq), then at 0° C. is added mesylchloride (34.8 ml, 0.44 mol, 1.3eq). The reaction mixture is stirred at 0° C. for 30 minutes and allowedto rise to room temperature. The reaction mixture is extracted withmethylene chloride (2×300 ml), the combined organic layers are thenwashed with brine (2×300 ml) and concentrated under vacuum. To the crudeproduct in solution in ethyl acetate (600 ml) is added sodium iodide(67.2 g, 0.44 mol, 1.3 eq) and the reaction mixture is stirred underreflux for 6 hours. After filtration, the organic layer is washed withbrine (3×400 ml), dried with Na₂SO₄, filtered and concentrated undervacuum. 1-Benzyloxy-4-(2-iodo-ethyl)-benzene is isolated aftercrystallization with diethyl ether (116.5 g, 86%).

To a solution of acetamidomalonate (59.4 g, 0.27 mol, 2 eq) in drydimethylformamide (400 ml) is added at 0° C. under inert atmospheresodium hydride (60% in oil) (9.94 g, 0.49 mol, 1.8 eq), the reactionmixture is stirred for 3 hours at 0° C.1-Benzyloxy-4-(2-iodo-ethyl)-benzene (46.8 g, 0.13 mol, 1 eq) insolution in dry dimethylformamide (250 ml) is then slowly added at 0° C.and the reaction mixture is stirred at room temperature overnight. Thereaction mixture is quenched with few drops of methanol and concentratedalmost to dryness under vacuum, then extracted with ethyl acetate andwashed subsequently with 1N HCl (2×500 ml), saturated solution of NaHCO₃(2×500 ml) and brine (2×500 ml), dried with Na₂SO₄, filtered andconcentrated under vacuum.2-Acetylamino-2-[2-(4-benzyloxy-phenyl)-ethyl]-malonic acid diethylester is isolated after multiple crystallization using diethyl ether(47.3 g, 80%).

To a solution of 2-acetylamino-2-[2-(4-benzyloxy-phenyl)-ethyl]-malonicacid diethyl ester (44.1 g, 0.1 mol) in ethanol water (2/1) (285 ml/285ml) is added CaCl₂ (28.5 g, 0.26 mol, 2.5 eq) and NaBH₄ by portion (19.4g, 0.52 mol, 5.0 eq), the reaction mixture is stirred overnight at roomtemperature. At 0° C. the reaction mixture is carefully quenched withdrop wise methanol (10 ml) and concentrated to almost dryness undervacuum. The crude mixture is extracted with ethyl acetate (4×500 ml) andwashed subsequently with 1N HCl (2×300 ml), saturated solution of NaHCO₃(2×300 ml) and brine (2×300 ml). The combined organic layers are thendried with Na₂SO₄, filtered and concentrated under vacuum.N-[3-(4-Benzyloxy-phenyl)-1,1-bis-hydroxymethyl-propyl]-acetamide iscarried on without further purification.

To a solution of crudeN-[3-(4-benzyloxy-phenyl)-1,1-bis-hydroxymethyl-propyl]-acetamide in amixture of tetrahydrofuran, methanol, water (1/2/2) (450 ml/900 ml/900ml) is added at room temperature lithium hydroxide (32.7 g, 1.36 mol,8.0 eq). The reaction mixture is stirred at 55° C. for 5 hours, thenextracted with ethyl acetate (500 ml) and washed with brine (2×300 ml),the combined organic layers are then dried with Na₂SO₄, filtered andconcentrated under vacuum.2-Amino-2-[2-(4-benzyloxy-phenyl)-ethyl]-propane-1,3-diol is isolatedafter crystallization using ethyl acetate (28.8 g, 97%).

To a solution of2-amino-2-[2-(4-benzyloxy-phenyl)-ethyl]-propane-1,3-diol (200 mg, 0.66mmol) in dioxane (10 ml) is added a solution of 1M NaOH (0.73 ml, 073mmol, 1.1 eq) and Boc anhydride (217 mg, 0.99 mmol, 1.5 eq). Thereaction mixture is then stirred overnight at room temperature.Extraction with ethyl acetate (80 ml) and washed with brine (2×50 ml),the combined organic layers are then dried with Na₂SO₄, filtered andconcentrated under vacuum.2-Amino-2-[2-(4-benzyloxy-phenyl)-ethyl]-propane-1,3-diol is isolated asa white solid after flash chromatography (Ethyl acetate/Hexane (3/1))and crystallization with diethyl ether (204 mg, 87%).

To a solution of2-amino-2-[2-(4-benzyloxy-phenyl)-ethyl]-propane-1,3-diol (6.31 g, 0.16mol) in dichloromethane (120 ml) and pyridine (1.26 ml, 0.16 mol) isadded at room temperature o-nitrobenzoylchloride (2.27 ml. 0.017 mol).The reaction mixture is then stirred overnight at room temperature.Extraction with dichloromethane (300 ml) and washed with brine (2×200ml), the combined organic layers are then dried with Na₂SO₄, filteredand concentrated under vacuum. 2-Nitro-benzoic acid4-(4-benzyloxy-phenyl)-2-tert-butoxycarbonylamino-2-hydroxymethyl-butylester is isolated as a white solid after flash chromatography (Ethylacetate/Hexane (1/1)) and crystallization with diethyl ether (6.55 mg,76%). 1.40 g of starting material could be isolated.

To a solution of 2-nitro-benzoic acid4-(4-benzyloxy-phenyl)-2-tert-butoxycarbonylamino-2-hydroxymethyl-butylester (105 mg, 0.19 mmol) in toluene (2 ml) is added 2.2dimethoxypropane (0.06 ml, 0.57 mmol, 3 eq) and a catalytic amount ofp-toluene sulfonic acid. The reaction mixture is stirred at 95° C. for 6hours, then concentrated to dryness under negative pressure.4-[2-(4-Benzyloxy-phenyl)-ethyl]-2,2-dimethyl-4-(2-nitro-benzoyloxy-methyl)-oxazolidine-3-carboxylicacid tert-butyl ester is isolated as an oil (88 mg, 78%) afterpurification by flash chromatography (Ethyl acetate/Hexane (1/3)).

To a solution of4-[2-(4-benzyloxy-phenyl)-ethyl]-2,2-dimethyl-4-(2-nitro-benzoyloxymethyl)-oxazolidine-3-carboxylicacid tert-butyl ester (80 mg, 0.13 mmol) in methanol (1 ml) and THF (1ml) is added K₂CO₃ (1.5 mg, 0.076 eq), the reaction mixture is stirredovernight at room temperature. The reaction mixture is concentrated todryness and4-[2-(4-benzyloxy-phenyl)-ethyl]-4-hydroxymethyl-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-butyl ester (51 mg, 85%) is isolated as a white solid afterflash chromatography (Ethyl acetate/Hexane (1/4)).

To a solution of4-[2-(4-benzyloxy-phenyl)-ethyl]-4-hydroxymethyl-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-butyl ester (25 mg, 0.05 mmol) in methanol (10 ml) is added acatalytic amount of paladium on charcoal (10% wt). The reaction mixtureis stirred under H₂ atmosphere at room temperature for 2 hours.4-Hydroxymethyl-4-[2-(4-hydroxy-phenyl)-ethyl]-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-butyl ester is isolated as a white solid after filtration ofthe suspension through celite and concentration to dryness.

To a solution of(S)-4-hydroxymethyl-4-[2-(4-hydroxy-phenyl)-ethyl]-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-butyl ester (100 mg, 0.28 mmol) in DMF (5 ml) is added CsCO₃(120.5 mg, 0.37 mmol, 1.3 eq) and 1-(2-bromo-ethoxy)-3-fluoro-benzene(80.7 mg, 0.37 mmol, 1.3 eq). The reaction mixture is stirred at 85° C.for 4 hours. Ethyl acetate and water are then added, the organic layeris separated and the aqueous phase is extracted with ethylacetate (3×50ml). The combined organic extracts are washed with brine, dried overMgSO₄, and evaporated to dryness. Purification by flash chromatography(AcOEt/Hx 9:1) affords(S)-4-(2-{4-[2-(3-fluoro-phenoxy)-ethoxy]-phenyl}-ethyl)-4-hydroxymethyl-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-butyl ester as colorless oil.

To a solution of(S)-4-(2-{4-[2-(3-fluoro-phenoxy)-ethoxy]-phenyl}-ethyl)-hydroxymethyl-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-butyl ester (70 mg, 0.14 mmol) and tetrazole (49 mg, 0.7 mmol,5 eq., recrystallized from toluene) in dry THF (5 ml) is addeddi-tBu-N,N-diisopropylphosphoramide (155 mg, 0.56 mmol, 4 eq.). Afterstirring under argon at RT for 3 h, H₂O₂ (30%, 10 eq.) is slowly addedat 0° C. with vigorous stirring. The reaction mixture is stirred forfurther 30 min, followed by addition of saturated sodium thiosulfatesolution (5 ml). The organic layer is separated and the aqueous phase isextracted with ether (3×20 ml). The combined organic extracts are washedwith brine, dried over MgSO₄, and evaporated to dryness. Purification byflash chromatography (AcOEt/Hx 1:1) affords(R)-4-(di-tert-butoxy-phosphoryloxymethyl)-4-(2-{4-[2-(3-fluoro-phenoxy)-ethoxy]-phenyl}-ethyl)-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester as colorless crystals.

Finally, a solution of(R)-4-(di-tert-butoxy-phosphoryloxymethyl)-4-(2-{4-[2-(3-fluoro-phenoxy)-ethoxy]-phenyl}-ethyl)-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-butyl ester (70 mg, 0.10 mmol) in conc. HCl (2 ml). Is stirredat room temperature for one hour and is then heated to 95° C. for 2hours, then concentrated to dryness. The residue is re-dissolved inethyl acetate and precipitated with hexanes. The solid is filtered off,washed with dry ether and dried in vacuo to afford phosphoric acidmono-((R)-2-amino-4-{4-[2-(3-fluoro-phenoxy)-ethoxy]-phenyl}-2-hydroxymethyl-butyl)esteras a colorless powder.

EXAMPLE 10 Phosphoric Acidmono-((S)-2-amino-4-{4-[2-(3-fluoro-phenoxy)-ethoxy]-phenyl}-2-hydroxymethyl-butyl)ester

Phosphoric acidmono-((S)-2-amino-4-{4-[2-(3-fluoro-phenoxy)-ethoxy]-phenyl}-2-hydroxymethyl-butyl)esteris prepared as described in example 9 using(R)-4-hydroxymethyl-4-[2-(4-hydroxy-phenyl)-ethyl]-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-butyl ester instead of(S)-4-hydroxymethyl-4-[2-(4-hydroxy-phenyl)-ethyl]-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-butyl ester.

EXAMPLE 11 Phosphoric Acidmono-{(R)-2-amino-2-hydroxymethyl-4-[4-(2-m-tolyloxy-ethoxy)-phenyl]-butyl}ester

Phosphoric acidmono-{(R)-2-amino-2-hydroxymethyl-4-[4-(2-m-tolyloxy-ethoxy)-phenyl]-butyl}esteris prepared using an analogous method to that described in example 9.

EXAMPLE 12 Phosphoric Acidmono-((R)-2-amino-2-hydroxymethyl-4-{4-[2-(4-methoxy-phenyl)-ethoxy]-phenyl}-butyl)ester

Phosphoric acidmono-((R)-2-amino-2-hydroxymethyl-4-{4-[2-(4-methoxy-phenyl)-ethoxy]-phenyl}-butyl)esteris prepared using an analogous method to that described in example 9.

EXAMPLE 13 Phosphoric Acidmono-{(R)-2-amino-2-hydroxymethyl-4-[4-(2-p-tolyl-ethoxy)-phenyl]-butyl}ester

Phosphoric acidmono-{(R)-2-amino-2-hydroxymethyl-4-[4-(2-p-tolyl-ethoxy)-phenyl]-butyl}esteris prepared using an analogous method to that described in example 9.

EXAMPLE 14 Phosphoric Acidmono-((R)-2-amino-2-hydroxymethyl-4-{4-[2-(4-trifluoromethyl-phenyl)-ethoxy]-phenyl}-butyl)ester

Phosphoric acidmono-((R)-2-amino-2-hydroxymethyl-4-{4-[2-(4-trifluoromethyl-phenyl)-ethoxy]-phenyl}-butyl)esteris prepared using an analogous method to that described in example 9.

EXAMPLE 15 Phosphoric Acid Diethyl Ester4-(2-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}-ethyl)-2-methyl-4,5-dihydro-oxazol-4-ylmethylEster

To a solution of phosphoric acidmono-(2-amino-4-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}-2-hydroxymethyl-butyl)ester(100 mg, 0.22 mmol) in triethyl orthoacetate (5 ml) is added acetic acid(13 ul, 0.22 mmol). The reaction mixture is stirred at 80° C. for 2hours and concentrated to dryness. Purification by flash chromatography(AcOEt/Hx 1:4) affords phosphoric acid diethyl ester4-(2-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}-ethyl)-2-methyl-4,5-dihydro-oxazol-4-ylmethylester as colorless oil.

EXAMPLE 16 Phosphoric Acid Diethyl Ester(S)-2-methyl-4-[2-(4-octyl-phenyl)-ethyl]-4,5-dihydro-oxazol-4-ylmethylEster

Phosphoric acid diethyl ester(S)-2-methyl-4-[2-(4-octyl-phenyl)-ethyl]-4,5-dihydro-oxazol-4-ylmethylester is prepared as described in example 15 using of phosphoric addmono-(2-amino-4-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}-2-hydroxymethyl-butyl)ester.

EXAMPLE 17 Phosphoric Acid Diethyl Ester(R)-2-methyl-4-[2-(4-octyl-phenyl)-ethyl]-4,5-dihydro-oxazol-4-ylmethylEster

Phosphoric acid diethyl ester(R)-2-methyl-4-[2-(4-octyl-phenyl)-ethyl]-4,5-dihydro-oxazol-4-ylmethylester is prepared as described in example 15 using phosphoric acidmono-(2-amino-4-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}-2-hydroxymethyl-butyl)ester.

EXAMPLE 18 Chromatographic Resolution of Racemic4-hydroxymethyl-4-[2-(4-hydroxy-phenyl)-ethyl]-2,2-dimethyl-oxazolidine-3-carboxylicAcid tert-butyl Ester

10 micro-liter of a 0.1% ethanol solution of racemic4-hydroxymethyl-4-[2-(4-hydroxy-phenyl)-ethyl]-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-butyl ester are injected on a Chiralcel OD-H column (0.46×25cm; commercially available from Chiral Technologies). Thechromatographic separation is achieved at room temperature and at a flowrate of 1 ml/min using a mixture of n-hexane/ethanol 90/10 (volume)containing 0.1% trifluoroacetic acid (TFA) as the mobile phase.Detection is performed by UV at 210 nm. The enantiomers eluterespectively after 7.23 min and 9.39 min (Separation factor α: 1.52)

EXAMPLE 19 Chromatographic Resolution of Racemic Phosphoric Acid DiethylEster4-(2-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}ethyl)-2-methyl-4,5-dihydro-oxazol-4-ylmethylEster

10 micro-liter of a 0.1% ethanol solution of racemic phosphoric aciddiethyl ester4-(2-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}ethyl)-2-methyl-4,5-dihydro-oxazol-4-ylmethylester are injected on a Chiralcel OD-H column (0.46×25 cm; commerciallyavailable from Chiral Technologies). The chromatographic separation isachieved at room temperature and at a flow rate of 1 ml/min using amixture of n-hexane/ethanol 95/5 (volume) as the mobile phase. Detectionis performed by UV at 210 nm. The enantiomers elute respectively after31.17 min and 35.44 min (Separation factor α: 1.15)

EXAMPLE 20 Chromatographic Separation of the Enantiomers Phosphoric AcidDiethyl Ester4-(2-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}ethyl)-2-methyl-4,5-dihydro-oxazol-4-ylmethylEster

10 micro-liter of a 0.1% ethanol solution of a mixture of theenantiomers phosphoric acid diethyl ester4-(2-{4-[2-(3-fluoro-4-methoxy-phenyl)-ethoxy]-phenyl}-ethoxyl)-2-methyl-4,5-dihydro-oxazol-4-ylmethylester are injected on a Chiralcel OJ-H column (0.46×25 cm; commerciallyavailable from Chiral Technologies). The chromatographic separation isachieved at room temperature and at a flow rate of 1 ml/min using amixture of n-hexane/2-propanol 75/25 (volume) as the mobile phase.Detection is performed by UV at 210 nm. The enantiomers eluterespectively after 13.89 min and 16.77 min (Separation factor α: 1.27)

EXAMPLE 21 Chromatographic Separation of the Enantiomers Phosphoric AcidDiethyl Ester(S)-2-methyl-4-[2-(4-octyl-phenyl)-ethyl]-4,5-dihydro-oxazol-4-ylmethylEster

10 micro-liter of a 0.1% ethanol solution of a mixture of theenantiomers phosphoric acid diethyl ester(S)-2-methyl-4-[2-(4-octyl-phenyl)-ethyl]-4,5-dihydro-oxazol-4-ylmethylester are injected on a Chiralpak AD-H column (0.46×25 cm; commerciallyavailable from Chiral Technologies). The chromatographic separation isachieved at room temperature and at a flow rate of 1 ml/min using amixture of n-hexane/ethanol 95/5 (volume) as the mobile phase. Detectionis performed by UV at 210 nm. The enantiomers elute respectively after10.35 min and 12.32 min (Separation factor α: 1.27).

The compounds of formula II comprising greater than 70% of the R or Senantiomer, especially the S enantiomer, in free form or inpharmaceutically acceptable salt form, (hereinafter referred to as thecompounds of the invention) exhibit valuable pharmacological properties,e.g. lymphocyte recirculation modulating properties, e.g. as indicatedin in vitro and in vivo tests and are therefore indicated for therapy.

A. In Vitro

The compounds of the invention have binding affinity to individual humanS1P receptors as determined in following assays:

Transient Transfection of Human S1P Receptors into HEK293 Cells

S1P receptors and G_(i) proteins are cloned, and equal amounts of 4cDNAs for the S1P receptor, G_(i)-α, G_(i)-β and G_(i)-γ are mixed andused to transfect monolayers of HEK293 cells using the calcium phosphateprecipitate method (M. Wigler et al., Cell. 1977; 11; 223 and DS. Im etal., Mol. Pharmacol. 2000; 57; 753). Briefly, a DNA mixture containing25 μg of DNA and 0.25 M CaCl is added to HEPES-buffered 2 mM Na₂HPO₄.Subconfluent monolayers of HEK293 cells are poisoned with 25 mMchloroquine, and the DNA precipitate is then applied to the cells. After4 h, the monolayers are washed with phosphate-buffered saline and refedmedia (90% 1:1 Dulbecco's modified essential media (DMEM):F-12+10% fetalbovine serum). The cells are harvested 48-72 h after addition of the DNAby scraping in HME buffer (in mM: 20 HEPES, 5 MgCl₂, 1 EDTA, pH 7.4)containing 10% sucrose on ice, and disrupted using a Dounce homogenizer.After centrifugation at 800×g, the supernatant is diluted with HMEwithout sucrose and centrifuged at 100,000×g for 1 h. The resultingpellet is rehomogenized and centrifuged a second hour at 100,000×g. Thiscrude membrane pellet is resuspended in HME with sucrose, aliquoted, andsnap-frozen by immersion in liquid nitrogen. The membranes are stored at70° C. Protein concentration is determined spectroscopically by Bradfordprotein assay.

GTPγS Binding Assay Using S1P Receptor/HEK293 Membrane Preparations

GTPγS binding experiments are performed as described by D S. Im et al.,Mol. Pharmacol. 2000; 57:753. Ligand-mediated GTPγS binding toG-proteins is measured in GTP binding buffer (in mM: 50 HEPES, 100 NaCl,10 MgCl₂, pH 7.5) using 25 μg of a membrane preparation from transientlytransfected HEK293 cells. Ligand is added to membranes in the presenceof 10 μM GDP and 0.1 nM [³⁵S]GTPγS (1200 Ci/mmol) and incubated at 30°C. for 30 min. Bound GTPγS is separated from unbound using the Brandelharvester (Gaithersburg, Md.) and counted with a liquid scintillationcounter.

In these assays, the compounds of the invention have binding affinitiesto S1P receptors in the sub-microM range.

In particular, the EC₅₀ values in nM for the following compounds atvarious S1P receptors are shown in the table below:

S1P-1 S1P-3 S1P-2 S1P-4 S1P-5 EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ nM nM nM nM nM(R)-FTY720-P 191.15 28.91 >10000 80.13 >10000 (S)-FTY720-P 0.333.21 >10000 0.75 0.33 Ex. 14 0.4 140 >10000 6.6 7.7wherein (R)-FTY720-P is (R)-FTY720-phosphate, (S)-FTY720-P is(S)-FTY720-phosphate, in contrast to (R)-FTY720-phosphate, which showsagonistic effects on S1P receptors only at very high concentrations,(S)-FTY720-phosphate is a full agonist on S1P1 and S1P3 and a partialagonist on S1P4 and S1P5 in the low nanomolar range.B. In Vivo: Blood Lymphocyte Depletion

A compound of the invention or the vehicle is administered orally bygavage to rats. Tail blood for hematological monitoring is obtained onday −1 to give the baseline individual values, and at 2, 6, 24, 48 and72 hours after drug application. In this assay, the compounds of theinvention deplete peripheral blood lymphocytes when administered at adose of 0.03 to 3 mg/kg.

The compounds of the invention are, therefore, useful in the treatmentand/or prevention of diseases or disorders mediated by lymphocytesinteractions, e.g. in transplantation, such as acute or chronicrejection of cell, tissue or organ allo- or xenografts or delayed graftfunction, graft versus host disease, autoimmune diseases, e.g.rheumatoid arthritis, systemic lupus erythematosus, hashimoto'sthyroids, multiple sclerosis, myasthenia gravis, diabetes type I or IIand the disorders associated therewith, vasculitis, pernicious anemia,Sjoegren syndrome, uveitis, psoriasis, Graves opthalmopathy, alopeciaagreata and others, allergic diseases, e.g. allergic asthma, atopicdermatitis, allergic rhinitis/conjunctivitis, allergic contactdermatitis, inflammatory diseases optionally with underlying aberrantreactions, e.g. inflammatory bowel disease, Crohn's disease orulcerative colitis, intrinsic asthma, inflammatory lung injury,inflammatory liver injury, inflammatory glomerular injury,atherosclerosis, osteoarthritis, irritant contact dermatitis and furthereczematous dermatitises, seborrhoeic dermatitis, cutaneousmanifestations of immunologically-mediated disorders, inflammatory eyedisease, keratoconjunctivitis, myocarditis or hepatitis,ischemia/reperfusion injury. e.g. myocardial infarction, stroke, gutischemia, renal failure or hemorrhage shock, traumatic shock, others,cancer, e.g. T cell lymphomas or T cell leukemias, infectious diseases,e.g. toxic shock (e.g. superantigen induced), septic shock, adultrespiratory distress syndrome or viral infections, e.g. AIDS, viralhepatitis or chronic bacterial infection. Examples of cell, tissue orsolid organ transplants include e.g. pancreatic islets, stem cells, bonemarrow, corneal tissue, neuronal tissue, heart, lung, combinedheart-lung, kidney, liver, bowel, pancreas, trachea or oesophagus.

For the above uses the required dosage will of course vary depending onthe mode of administration, the particular condition to be treated andthe effect desired. In general, satisfactory results are indicated to beobtained systemically at daily dosages of from about 0.03 to 2.5 mg/kgper body weight. An indicated daily dosage in the larger mammal, e.g.humans, is in the range from about 0.5 mg to about 100 mg, convenientlyadministered, for example, in divided doses up to four times a day or inretard form. Suitable unit dosage forms for oral administration comprisefrom ca. 1 to 50 mg active ingredient.

The compounds of the invention may be administered by any conventionalroute, in particular enterally, e.g. orally, e.g. in the form of tabletsor capsules, or parenterally, e.g. in the form of injectable solutionsor suspensions, topically, e.g. in the form of lotions, gels, ointmentsor creams, or in a nasal or a suppository form. Pharmaceuticalcompositions comprising a compound of the invention in free form or inpharmaceutically acceptable salt form in association with at least onepharmaceutical acceptable carrier or diluent may be manufactured inconventional manner by mixing with a pharmaceutically acceptable carrieror diluent.

The compounds of the invention may be administered in free form or inpharmaceutically acceptable salt form e.g. as indicated above. Suchsalts may be prepared in conventional manner and exhibit the same orderof activity as the free compounds.

In accordance with the foregoing the present invention further provides:

-   1.1 A method for preventing or treating disorders or diseases    mediated by lymphocytes, e.g. such as indicated above, in a subject    in need of such treatment, which method comprises administering to    said subject an effective amount of a compound of formula II    comprising greater than 70% by weight of the R or S enantiomer, or a    pharmaceutically acceptable salt thereof;-   1.2 A method for preventing or treating acute or chronic transplant    rejection or T-cell mediated inflammatory or autoimmune diseases,    e.g. as indicated above, in a subject in need of such treatment,    which method comprises administering to said subject an effective    amount of a compound of formula II comprising greater than 70% by    weight of the R or S enantiomer, or a pharmaceutically acceptable    salt thereof;-   2. A compound of formula II comprising greater than 70% by weight of    the R or S enantiomer, in free form or in a pharmaceutically    acceptable salt form for use as a pharmaceutical, e.g. in any of the    methods as indicated under 1.1 or 1.2 above.-   3. A pharmaceutical composition, e.g. for use in any of the methods    as in 1.1 or 1.2 above comprising a compound of formula II    comprising greater than 70% by weight of the R or S enantiomer in    free form or pharmaceutically acceptable salt form in association    with a pharmaceutically acceptable diluent or carrier therefor.-   4. A compound of formula II comprising greater than 70% by weight of    the R or S enantiomer or a pharmaceutically acceptable salt thereof    for use in the preparation of a pharmaceutical composition for use    in any of the method as in 1.1 or 1.2 above.

The compounds of the invention may be administered as the sole activeingredient or in conjunction with, e.g. as an adjuvant to, other drugse.g. immunosuppressive or immunomodulating agents or otheranti-inflammatory agents, e.g. for the treatment or prevention of allo-or xenograft acute or chronic rejection or inflammatory or autoimmunedisorders, or a chemotherapeutic agent, e.g a malignant cellanti-proliferative agent. For example, the compounds of the inventionmay be used in combination with a calcineurin inhibitor, e.g.cyclosporin A or FK 506; a mTOR inhibitor, e.g. rapamycin,40-O-(2-hydroxyethyl)-rapamycin, CCI779 or ABT578; an ascormycin havingimmunosuppressive properties, e.g. ABT-281, ASM981, etc.;corticosteroids; cyclophosphamide; azathioprene; methotrexate,leflunomide; mizoribine; mycophenolic acid; mycophenolate mofetil;15-deoxyspergualine or an immunosuppressive homologue, analogue orderivative thereof; immunosuppressive monoclonal antibodies, e.g.,monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4,CD7, CD8, CD25, CD28, CD40. CD45, CD58, CD80, CD86 or their ligands;other immunomodulatory compounds, e.g. a recombinant binding moleculehaving at least a portion of the extracellular domain of CTLA4 or amutant thereof, e.g. an at least extracellular portion of CTLA4 or amutant thereof joined to a non-CTLA4 protein sequence, e.g. CTLA4Ig (forex. designated ATCC 68629) or a mutant thereof, e.g. LEA29Y; adhesionmolecule inhibitors, e.g. LFA-1 antagonists, ICAM-1 or -3 antagonists,VCAM-4 antagonists or VLA-4 antagonists; or a chemotherapeutic agent,e.g. paclitaxel, gemcitabine, cisplatinum, doxorubicin or5-fluorouracil.

Where the compounds of the invention are administered in conjunctionwith other immunosuppressive/immunomodulatory, anti-inflammatory orchemotherapeutic therapy, dosages of the co-administeredimmunosuppressant, immunomodulatory, anti-inflammatory orchemotherapeutic compound will of course vary depending on the type ofco-drug employed, e.g. whether it is a steroid or a calcineurininhibitor, on the specific drug employed, on the condition being treatedand so forth. In accordance with the foregoing the present inventionprovides in a yet further aspect:

-   5. A method as defined above comprising co-administration, e.g.    concomitantly or in sequence, of a therapeutically effective    non-toxic amount of a compound of the invention and at least a    second drug substance, e.g. an immunosuppressant, immunomodulatory,    anti-inflammatory or chemotherapeutic drug, e.g. as indicated above.-   6. A pharmaceutical combination, e.g. a kit, comprising a) a first    agent which is a compound of the invention as disclosed herein, in    free form or in pharmaceutically acceptable salt form, and b) at    least one co-agent, e.g. an immunosuppressant, immunomodulatory,    anti-inflammatory or chemotherapeutic drug. The kit may comprise    instructions for its administration.

The terms “co-administration” or “combined administration” or the likeas utilized herein are meant to encompass administration of the selectedtherapeutic agents to a single patient, and are intended to includetreatment regimens in which the agents are not necessarily administeredby the same route of administration or at the same time.

The term “pharmaceutical combination” as used herein means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. a compound of the invention and a co-agent, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g. a compound of the invention and a co-agent, are bothadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the 2compounds in the body of the patient. The latter also applies tococktail therapy, e.g. the administration of 3 or more activeingredients.

In a further aspect, the present invention provides a method ofseparating the R and S enantiomers of a compound of formula I,comprising separating the enantiomers by high performance liquidchromatography (HPLC) using a chiral ion-exchange phase e.g. based onquinine carbamate or quinidine carbamate as described above.

In a further aspect, the present invention provides a method ofdetermining the amount of the R and/or S isomers of a compound offormula II present in a sample, comprising (a) reacting the compound offormula II present in the sample with benzene-1,2-dicarbaldehyde to forma compound of formula I, and separating the R and S isomers of thecompound of formula I by HPLC. The HPLC is preferably performed asdescribed above, using a chiral ion-exchange phase e.g. based on quininecarbamate or quinidine carbamate. The compounds of formula I are usefulas intermediates in such a method. The sample may be e.g. a samplederived from a bodily fluid e.g. blood, plasma, saliva, or urine, andmay be first subjected to one or more separation steps, (e.g. extractionwith methanol and/or non-chiral HPLC) in order to separate the compoundof formula II from the fluid. By such a method the amount of the activeR or S enantiomer in the sample may be determined. Thus the method maybe useful e.g. for monitoring the blood concentration of the active Senantiomer of a compound of formula II in a subject, followingadministration of a compound of formula II (e.g. a racemic mixturethereof) or following administration of a precursor of a compound offormula II (forming the compound of formula II as a metabolite in thebody) to the subject.

For example, achiral 14C-labeled FTY720 is administered to rats eitherorally (7.5 mg/kg) or by intravenous infusion (4 mg/kg). Chiral14C-labeled FTY720-phosphate is formed in the body of the rat as ametabolite of 14C-labeled FTY720. Blood samples are taken at differenttimes (e.g. 3 or 72 hours) after dosing. Each blood sample is extractedwith methanol and [14C]FTY720-phosphate is isolated by nonchiral HPLC.The isolated [14C]FTY720-phosphate is derivatized with OPA. Thederivative is spiked with unlabelled OPA-derivatized R and SFTY720-phosphate as retention time markers (monitored by UV at 215 nm)and subjected to chiral HPLC separation using a ProntoSIL Chiral AX QN-1column. The [14C]FTY720-phosphate in all blood samples representsexclusively the pharmacologically active S-enantiomer. The inactiveR-enantiomer is not detectable.

1. A process for producing a compound of the formula II

wherein each of m and n, independently, is 1, 2 or 3 X is O or a directbond; R₁ is a phenylalkyl wherein alkyl is a straight- or branched(C₆₋₂₀)carbon chain; or a phenylalkyl wherein alkyl is a straight- orbranched (C₁₋₃₀)carbon chain wherein said phenylalkyl is substituted atthe phenyl residue by a straight- or branched (C₆₋₂₀)carbon chainoptionally substituted by halogen, a straight- or branched (C₆₋₂₀)alkoxychain optionally substituted by halogen, a straight- or branched(C₆₋₂₀)alkenyloxy, phenylalkoxy, halophenylalkoxy, phenylalkoxyalkyl,phenoxyalkoxy or phenoxyalkyl, cycloalkylalkyl substituted byC₆₋₂₀alkyl, heteroarylalkyl substituted by C₆₋₂₀alkyl, heterocyclicC₆₋₂₀alkyl or heterocyclic alkyl substituted by C₂₋₂₀alkyl, and whereinthe alkyl moiety may have in the carbon chain, a bond or a heteroatomselected from a double bond, a triple bond, O, S, sulfinyl, sulfonyl, orNR₅, wherein R₅ is H, alkyl, aralkyl, acyl or alkoxycarbonyl, and as asubstituent alkoxy, alkenyloxy, alkynyloxy, aralkyloxy, acyl,alkylamino, alkylthio, acylamino, alkoxycarbonyl, alkoxycarbonylamino,acyloxy, alkylcarbamoyl, nitro, halogen, amino, hydroxy or carboxy, andR₂ is

wherein each of R₃ and R₄, independently, is H or C₁₋₄alkyl, whereinalkyl optionally substituted by 1, 2 or 3 halogen atoms; or apharmaceutically acceptable salt thereof; and wherein greater than 70%of the compound by weight is in the form of the R or S enantiomer;comprising deprotecting a compound of formula III, IIIa or IIIb whereingreater than 70% by weight of the compound of formula III, IIIa or IIIbis in the form of the R or S enantiomer

wherein n, m, X, R₁ and R₂ are as defined above, R₆ is an aminoprotecting group, and R₆′, is an amino protecting group such that R₆′,together with the oxygen and nitrogen atoms to which it is attached, theasymmetric carbon atom and 1 to 3 additional carbon atoms forms a 5 to 7membered heterocyclic ring; and, where required, converting thecompounds of formula II obtained in free form into the salt form, orvice versa.
 2. The process according to claim 1 wherein the compound offormula II is phosphoric acidmono-[2-amino-2-hydroxymethyl-4-(4-octyl-phenyl)-butyl]ester orphosphoric acidmono-[2-hydroxymethyl-4-(4-octyl-phenyl)-2-(1-oxo-1,3-dihydro-isoindol-2-yl)-butyl]ester.3. A process for obtaining a compound of formula III, IIIa or IIIb, asdefined in claim 1, wherein greater than 70% of the compound by weightis in the form of the R or S enantiomer, comprising separating the Senantiomer from the R enantiomer in a racemic mixture of a compound offormula III, IIIa or IIIb using chromatography or simulated moving bedchromatography with a polysaccharide-based chiral stationary phase. 4.The process according to claim 3 wherein the stationary phase isamylose-type phase.
 5. A process for obtaining a compound of formulaIII, IIIa or IIIb, as defined in claim 1, wherein greater than 70% ofthe compound by weight is in the form of the R or S enantiomer,comprising the steps of i) obtaining a compound of formula IV, IVa orIVb, wherein greater than 70% of the compound by weight is in the formof the R or S enantiomer

wherein each of m and n, independently, is 1, 2 or 3 X is O or a directbond R₁ is a phenylalkyl wherein alkyl is a straight- or branched(C₆₋₂₀)carbon chain; or a phenylalkyl wherein alkyl is a straight- orbranched (C₁₋₃₀)carbon chain wherein said phenylalkyl is substituted atthe phenyl residue by a straight- or branched (C₆₋₂₀)carbon chainoptionally substituted by halogen, a straight- or branched (C₆₋₂₀)alkoxychain optionally substituted by halogen, a straight- or branched(C₆₋₂₀)alkenyloxy, phenylalkoxy, halophenylalkoxy, phenylalkoxyalkyl,phenoxyalkoxy or phenoxyalkyl, cycloalkylalkyl substituted byC₆₋₂₀alkyl, heteroarylalkyl substituted by C₆₋₂₀alkyl, heterocyclicC₆₋₂₀alkyl or heterocyclic alkyl substituted by C₂₋₂₀alkyl, and whereinthe alkyl moiety may have in the carbon chain, a bond or a heteroatomselected from a double bond, a triple bond, O, S, sulfinyl, sulfonyl, orNR₅, wherein R₅ is H, alkyl, aralkyl, acyl or alkoxycarbonyl, and as asubstituent alkoxy, alkenyloxy, alkynyloxy, aralkyloxy, acyl,alkylamino, alkylthio, acylamino, alkoxycarbonyl, alkoxycarbonylamino,acyloxy, alkylcarbamoyl, nitro, halogen, amino, hydroxy or carboxy, andR′₂ is

wherein each of R₃′ and R₄′ is a hydrolysable group and optionally formtogether a cyclic system, R₆ is an amino protecting group, and R₆′ is anamino protecting group such that R₆′, together with the oxygen andnitrogen to which it is attached, the asymmetric carbon atom and from 1to 3 additional carbon atoms, forms a 5 to 7 membered heterocyclic ring;and ii) removing the hydrolysable groups present in R′₂.
 6. The processaccording to claim 5 wherein R₃′ and R₄′ form together a cyclic system.7. A process according to claim 1 wherein R₆′ is —C(O)— and the compoundthat is deprotected is a compound of formula IIIa.
 8. A processaccording to claim 5 wherein R₆′ is —C(CH₃)— and the compound that isdeprotected is a compound of formula IIIb.